Bifunctional Peptidylglycine α-Amidating Enzyme Requires 2 Copper Atoms for Maximum Activity

oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the Cu H center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.

mentioning

confidence: 99%

The Hydrogen Peroxide Reactivity of Peptidylglycine Monooxygenase Supports a Cu(II)-Superoxo Catalytic Intermediate

Bauman

Yukl

Alkevich

et al. 2006

“…The hydroxylation reaction catalyzed by DBM requires copper, reduced ascorbate, and molecular oxygen (Fig. 1A) (5)(6)(7). Neuropeptides are synthesized from larger inactive precursors that undergo a series of post-translational modifications, all of which occur within the secretory pathway lumen (8,9).…”

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confidence: 99%

Cell Type-specific Storage of Dopamine β-Monooxygenase

Oyarce

Eipper

2000

Expression of dopamine ␤-monooxygenase (DBM), the enzyme that converts dopamine into norepinephrine, is limited to adrenal chromaffin cells and a small population of neurons. We studied DBM trafficking to regulated granules by stably expressing rat DBM in AtT-20 corticotrope tumor cells, which contain regulated granules, and in Chinese hamster ovary (CHO) cells, which lack regulated granules. The behavior of exogenous DBM in both cell lines was compared with endogenous DBM in adrenal chromaffin cells. CHO cells secreted active DBM, indicating that production of active enzyme does not require features unique to neuroendocrine cells. Pulse-chase experiments indicated that early steps in DBM maturation followed a similar time course in AtT-20, CHO, and adrenal chromaffin cells. Use of a conformation-sensitive DBM antiserum indicated that acquisition of a folded structure occurred with a similar time course in all three cell types. Cell type-specific differences in DBM trafficking became apparent only when storage in granules was examined. As expected, DBM was stored in secretory granules in chromaffin cells; CHO cells failed to store DBM. Despite the fact that AtT-20 cells have regulated granules, exogenous DBM was not stored in these granules. Thus storage of DBM in secretory granules requires cell type specific factors.

“…Since PAM requires both ascorbate and copper for activity (33,34), several investigators have demonstrated changes in the production of amidated peptides by altering the levels of these cofactors in vivo. For example, a diet deficient in vitamin C has been shown to cause a 30-fold increase in the levels of glycine-extended gastrin with a concomitant 2-fold decrease in gastrin in extracts of guinea pig antra (35).…”

Section: Discussionmentioning

confidence: 99%

Suppression of Substance P Biosynthesis in Sensory Neurons of Dorsal Root Ganglion by Prodrug Esters of Potent Peptidylglycine α-Amidating Monooxygenase Inhibitors

Jeng

Fujimoto

Chou

et al. 1997

Substance P as well as many other neuropeptides are synthesized as glycine-extended precursors and converted to the biologically active C-terminal amides by posttranslational modification. The final step of posttranslational processing is catalyzed by peptidylglycine ␣-amidating monooxygenase (PAM). In a previous study, N-substituted homocysteine analogs were found to be potent inhibitors of PAM partially purified from conditioned medium of cultured rat medullary thyroid carcinoma CA-77 cells. These compounds, however, were only modest inhibitors of substance P production in cultured dorsal root ganglion cells, possibly because of poor cell penetration. Several ester derivatives of hydrocinnamoyl-phenylalanyl-homocysteine, one of the most potent PAM inhibitors, were prepared to increase the intracellular accessibility of these compounds. Hydrocinnamoyl-phenylalanyl-(S-benzoyl-homocysteine) benzyl ester was identified as the most potent compound, inhibiting substance P biosynthesis in dorsal root ganglion cells with an IC 50 of 2 M. Inhibition of PAM resulted in a concomitant increase in the glycine-extended substance p (substance P-Gly) precursor peptide. In the presence of 3 M benzyl ester derivative, the intracellular substance P-Gly level was 2.4-fold higher while the substance P level was 2.1-fold lower than the corresponding peptides in control cells. These results suggest that PAM inhibition represents an effective method for suppression of substance P biosynthesis and, therefore, may have therapeutic utility in conditions associated with elevated substance P levels. Furthermore, PAM inhibition may also prove useful in decreasing other amidated peptides.The C-terminal amide is a prerequisite for full biological activity of many neuropeptides (1). These neuropeptides are typically synthesized as glycine-extended precursors and converted to the mature peptides by a family of enzymes involved in posttranslational modifications, e.g. O-glycosylation, phosphorylation, sulfation, and hydroxylation, as well as in proteolytic processing, e.g. endoproteolysis and exoproteolysis (2). The final step of posttranslational processing is catalyzed by an enzyme originally identified as peptidylglycine ␣-amidating monooxygenase (PAM) 1 (3-5). PAM is localized in secretory granules and requires copper, ascorbate, and molecular oxygen for activity (2, 6, 7). Recent studies have shown that PAM is actually a bifunctional enzyme that contains two distinct enzymatic activities and catalyzes the C-terminal amidation in a sequential manner. The first enzyme, peptidylglycine ␣-hydroxylating monooxygenase (PHM) (EC 1.14.17.3), requires the cofactors for activity and catalyzes the peptidylglycine ␣-hydroxylation reaction while the second enzyme, peptidyl ␣-hydroxyglycine ␣-amidating lyase (PAL) (EC 4.3.2.5), converts the intermediate into an ␣-amidated peptide and glyoxylate (8 -11). Among the glycine-extended neuropeptides examined, glycine-extended substance P (substance P-Gly) has been demonstrated to possess the highest affinity ...